The goal of this application is to identify plasma peptide signatures that reflect neuroplasticity in regions of the brain associated with multiple stages of the addictive process, modeled initially using the simple conditioned place preference (CPP) procedure in rats. A focused approach is proposed in which the initial search will begin in the extracellular compartment of brain regions implicated in addictive processes, using microdialysis linked to mass spectrometry (high resolution nanobore LC-MS and data-dependent LC-MS2). We will then search for, and quantify, such identified candidate peptides in plasma in a targeted fashion, using LC-MS3 and/or LC-MRM. The discovery phase of the project will identify arrays of candidate peptides that, in brain regions considered central to addictive behavior, change extracellularly in response to chronic exposure to, and abstinence from, two highly addictive drugs of different pharmacological classes;namely morphine and cocaine. A parallel strategy will focus entirely on readily releasable peptides by constructing a subtractive screen of brain dialysates under potassium depolarization versus basal conditions. In both of these approaches blood plasma samples will be collected simultaneously. Peptides identified in either of these dialysate screens will subsequently be searched for in plasma in a targeted fashion. Peptides that are (a) detectable in both dialysate and plasma, and (b) change in concentration in tandem as a product of drug history or depolarization will be selected for the validation phase. In the validation phase, candidates identified in the discovery phase will be studied in longitudinal experiments using the place preference paradigm to correlate changes in plasma content with multiple phases of the conditioning experiment: drug conditioning, initial testing, incubation (withdrawal), re-testing, extinction and reinstatement. We expect to identify individual peptide signatures in plasma that reflect different stages of the addiction process to the extent that they are modeled by CPP. These experiments will focus on cocaine and morphine, but will be extended to other abused drugs and to other behavioral models (e.g. self-administration) in future experiments beyond the scope of this proposal. PUBLIC HEALTH RELEVANCE: These studies will provide plasma peptide signatures unique to different stages of one simple model of addictive behavior. Once this proof of principle is established the approach could be extended to other more complex addiction models, and ultimately to humans, thereby providing markers of underlying addictive processes. This is a key issue for the development of effective medications to treat addiction to multiple classes of abused substances.